The goal of this research proposal is to characterize structural features of the human lutropin/chorionic gonadotropin receptor (LHR) that are involved in signal transduction. The LHR, a member of the G protein-coupled receptor family, plays a key role in normal and abnormal reproductive physiology, but little is known about the specific mechanism by which it and other glycoprotein hormone receptors transmit signals across the cell membrane. The membrane-spanning domain of the LHR consists of a bundle of seven alpha-helical segments. Hormone binding to the extracellular surface triggers a conformational rearrangement in interhelical packing that allows the cytoplasmic face of the LHR to bind and activate G proteins. Many of the physiological effects of LH and hCG are mediated by Gs, the G protein that activates adenylyl cyclase and leads to increased intracellular cAMP, but additional signaling pathways may be involved in stimulating ovulation and luteinization and controlling growth of gonadal cells. The identity of the G proteins that mediate these effects is unknown, but Gq, a G protein that activates phospholipase C (PLC), is a likely candidate. The location of disease-associated and artificially generated mutations in the human LHR has helped identify conserved structural regions that may be involved in receptor activation and G protein coupling. In this proposal, site-directed mutagenesis, molecular modeling, and NMR-based structural studies of helical segments of the human LHR will be used to identify key intrahelical and interhelical bonds that are responsible for maintaining the inactive receptor conformation. An in vitro reconstitution assay will be used to quantitatively assess the ability of normal and mutant LHR to bind and activate different classes of G proteins that may be involved in ovarian cell signaling. It is anticipated that these studies will provide important insights into the mechanisms by which human LHR signaling affects ovarian function in health and disease.